On account of renewed interest in triple-stranded nucleic acid complexes we are carrying out a comprehensive thermodynamic study of helix formation among DNA homopolymers containing adenine, thymine and uracil. We have found that dissociation of a third strand of poly(dT) vs. that of poly(dU) from the same poly(dA).poly(dT) double helix over the range of 35 degrees C to 75 degrees C is characterized by a 400 cal/mol greater enthalpy change that is offset by a 1 cal/deg/mol entropy change. The heat capacity change in the reaction was found to be the same for the two different polymer systems. Since the heat capacity change is the hallmark characteristic of the hydrophobic effect, this result indicates that there is little change in solvent exposure of the thymine methyl groups upon dissociation of the third polypyrimidine strand. Since the structures of the two complexes are very likely identical, these experiments establish the magnitude of the energy changes in a well defined comparison between poly(dT) and poly(dU).